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UDC 616.832-004.2: 612.017.1: 575.174.015.3
THE CONNECTION BETWEEN IMMUNE SYSTEM GENES AND SPECIAL FEATURES OF THE COURSE OF MULTIPLE SCLEROSIS
1 Altai State Medical University, Barnaul
2 Regional clinical hospital, Barnaul
I.V. Smaginai, S.A. Yelchaninovai, Yu.V. Bodrova2
In this study we assessed the connection between HLA-DRB1, TNFa (rs1800629), TNFRSF1A (rs4149584), CD40 (rs6074022, rs11086998) and the course of multiple sclerosis (MS) in 100 relapsing-remitting multiple sclerosis (MS) patients who were Caucasian native-born residents of Altai Krai (Russia). The association of G/A TNFRSF1A (rs4149584) with a high neurologic deficit progression rate and also the connection between HLA-DRB1*3 and disease frequent relapses were revealed. Key words: multiple sclerosis, relapse, progression, genotype.
Multiple sclerosis (MS) is a chronic multifactorial demyelinating central nervous system disease characterized by a disability of mainly working-age patients (Shmidt T.Ye., Yakhno N.N., 2012).
According to modern theories the MS pathogenesis is based on central nervous system autoimmune inflammation accompanied by axon demyelination during the relapse period and by partial axon remyelination during the remission period and also on oligodendrocyte apoptosis leading to axon death and to irreversible disorders of nervous impulse conduction (Gusev Ye.I., Zavalishin I.A., Boyko A.N., 2011; Lassman H., 2014). In the case of the most widely-spread relapsing-remitting MS type the first years of the disease are characterized by the predominance of autoimmune inflammation with relapses followed by periods of remission (often partial), and the later years are characterized by neurodegeneration with rapid and irreversible development of multiple sclerosis neurological symptoms (Shmidt T.Ye., Yakhno N.N., 2012; Lassman H., 2014).
The frequency of MS relapses and MS progression rate widely vary (Stadelmann Ch., 2008; Tremlett H. et al., 2008). The reasons and mechanisms of these varieties in the MS course in different patients are not yet revealed, although this issue is the object of the majority of MS studies in recent years. In the modern period of studying mechanisms determining special features of the MS course pathogenetic determinants of MS relapses and neurological disorders progression are being revealed. It is believed that the MS type is determined by patient's genetic features which are most likely formed by polymorphic variants of immune system genes, protein products of which are involved into the pathogenesis of this disease and in some populations are the risk factors for this disease (Smagina I.V. et al., 2011; Favorova O.O., Kulakova O.G., Boyko A.N., 2010). In the first place these are HLА-system genes, ligand genes and genes of tumour necrosis factor (TNF) receptors and genes of other cytokines - immune response regulators. The results of this hypothesis testing are largely contradictory (Sаwсer S., 2009). This is due to the fact that the occurrence of gene polymorphism, the range and influence power of MS risk factors and, consequently, associated traits of genome special features significantly differ in various populations (Fоrte G.I. et аl., 2006; Ristk S.
еt аl., 2007). The most evidentiary are the facts that indicate the correlation of a 'light' MS course with female sex (Gusev Ye.I., Zavalishin I.A., Boyko A.N., 2011).
The revealing of the most significant factors of MS progression is necessary for the design of principles for reasonable use of MS disease modifying drugs (MSDMD). These widely used expensive drugs can prevent relapses and can slow neurologic deficit progression rate in 30% of patients when being prescribed at an early stage of MS (Smidt T.Ye., Yakhno N.N., 2012). There are reasons to believe that the improper effectiveness of these drugs is largely determined by the empiric way in which certain drugs of this pharmacological group are chosen and also by the switch to more aggressive therapy only after a months-long follow-up period (Grоssmаn I. et аl., 2007; Mаhurкаr S. еt аl., 2013).
Study purpose: to assess the connection between polymorphism of immune system genes and MS progression rate.
Subjects and methods.
100 MS patients were included into this randomized retrospective study. All studied patients had the following phenotypic characters in common - they all were Caucasian native-born residents of Altai Krai who had never lived in other regions before the study. The group was randomly formed from the population of MS patients in Altai Krai and included 10% of the total number of MS patients (1001 patients, as of 1st January 2012). The study inclusion criteria were: relapsing-remitting type of MS, disease duration of no less than 5 years, disability characterized with no more than 6.5 points according to the Exрanded Disability Status Sсale (EDSS) (Kurtzre J.F., 1983). The exclusion criteria of the study were: any autoimmune diseases except MS; administration of MS disease modifying drugs (MSDMD) and stem cell therapy before the study.
Patients with the disease duration of no less than 5 years were included into the study because during the MS initial period the dynamics of neurologic deficit progression rate usually largely varies. This is probably due to individual peculiarities of combination of immune inflammation fading with the course of the disease and of boosting of central nervous system degeneration process. After the
development of serious irreversible neurological disorders (EDSS more than 6.5) which quite often persist for many years, a slow disability progression rate does not indicate a good variant of MS course.
MS was diagnosed according to the McDonald criteria (Polman C.H. et al., 2005). Magnetic resonance imaging of the brain and spine was performed with Impact tomographic scanner (Siemens-Magnetom, Japan), magnetic field intensity of 1T. Standard T1- and T2-imaging and TIRM were used. Intravenous injections of gadolinium-containing drug Gadovist (Bayer Schering Pharma, FRG) in a dose of 0.1-0.3 mole/kg were made for contrasting purposes.
MS progression rate (PR) was calculated as the ratio of EDSS at the moment of examination to disease duration (Malkova N.A., 2006; Verians E. et al., 1983). In order to analyze the connection between MS PR and gene polymorphism the patients were divided into three groups: patients with a slow PR (<0.25 points/year) - 13 people (13%), patients with a medium PR (0.25<PR<0.75 points/year) - 64 people (64%) and patients with a high PR (>0,75 points/year) - 23 people (23%). The characteristics of the studied patients is represented in Table 1.
Characteristics of MS Patients
Prism 310 Genetic Analyzer together with BigDye Terminator Cycle Sequencing Ready Reaction Kit (PerKin Elmer, USA) following manufacturer's instructions. Sagner sequencing was performed using Eppendorf Mastercycler gradient amplifier (Eppendorf, USA).
We used Statistica v. 6.0 to compare the groups with the help of the two-tailed Fisher's exact test and Mann-Whitney U test, to assess the correlations between the variables with the help of the Spearman's rank correlation coefficient (rs) and to assess the odds ratio with the help of the logistic regression analysis. The genotypes deviation from Hardy-Weinberg equilibrium was evaluated using chi-square method with the help of the DeFinetti programme available on the Institute of Human Genetics (Munich, FRG) official website (https:// ihg.helmholtz-muenchen.de/cgi-bin/hw/hwa1. pl). The frequency of all genotypes matched with Hardy-Weinberg equilibrium (p>0.2). The results of the certain MS alleles association analysis are given as the additive model of inheritance which is best matched to the acquired data according to the Akaike information criterion value. The results for quantitative variables are specified as sample mean (M) and standard deviation (±SD), in some
Table 1
Included in the Study (n=100)
Parameter
Age, years (M ± SD) 36,8 ± 11,3
Male : female 24:76
Age of the MS onset, years (M ± SD) 27,4 ± 9,1
Disease duration, years (M ± SD) 7,8 ± 1,6
Disability according to EDSS, points (M ± SD) 3,4 ± 1,7
MS progression rate, points/year 0,50 ±0,22
First remission duration, months (M ± SD) 38,8 ± 24,4
The genotyping assay was performed for HLA-DRB1, TNFa (rs1800629), TNFRSF1A (rs4149584), CD40 (rs6074022, rs11086998). Выбор для исследования HLA-DRB1, однонуклеотидных полиморфизмов генов TNFa, TNFRSF1A, CD40 проводили с учетом данных об их связи с риском развития и/или патогенезом аутоиммунных заболеваний, включая РС (OKsenberg J.R et al., 2010). The selection of HLA-DRB1, single nucleotide polymorphisms of genes TNFa, TNFRSF1A, CD40 was made with consideration for the data about their connection with the risk of development and/or with the pathogenesis of autoimmune diseases including MS (Oksenberg J.R et al., 2010). DNA was recovered from the venous blood using the standard procedure including blood cells recovery and lysis, protein hydrolysis using protease K, DNA cleaning using phenolchloroform and ethanol precipitation of DNA. Genotyping was performed using TaqMan probes and iCyder iQ5 amplifier (Вю-Rad, USA). During the genotyping of HLA-DRB1 the sequencing of amplified DNA fragments after their desalting using gel filtration through an Acrilex P-10 column (Reanal, Hungary) was performed according to Sagner technology using automatic sequenator ABI
cases as 95% confidence interval (CI). The level of significance for all statistical criteria is p < 0,05.
The study was approved by the Ethics Commitee of Federal State Budgetary Educational Institution of Higher Education Altai State Medical University of the Ministry of Health of Russia.
Results and discussion. According to the results of genotyping the risk of a high MS progression rate is connected only with one of the studied gene specificity - TNFRSF1A (rs4149584). However, genotype G/A TNFRSF1A (rs4149584) is associated with a higher risk of a high MS PR whereas genotype G/G, on the contrary, is associated with a low disability progression rate (Table 2). No associations of HLA-DRB1 genotypes (including MS risk genotypes in Altai Krai) with a high MS PR were revealed. These are DRB1*3 genotype (heterozygous state) and DRB1*15 genotype (homo- and heterozygous state) (Smagina I.V. et al., 2011).
During the study of gender-based features of MS patients disability progression rate we revealed that a PR is twice higher in male patients than in female patients (0.74±0.23 and 0.37±0.05 points a
Table 2
Relative Risk of a High Multiple Sclerosis Progression Rate Depending on Genotypes TNFa (rs1800629), TNFRSF1A (rs4149584), CD40 (rs6074022, rs11086998)
Polymorphism genotype Genotype frequency, % Odds ratio, mean value (CI) Level of significance, p
Low and medium PR High PR
TNFa (rsl800629)
G/G 56 21 1,14 (0,42-3,11) 0,791
G/A 15 7 1,04 (0,37-2,92) 0,942
A/A 0 1 NA NA
TNFRSF1A (rs4149584)
G/G 59 26 0,05 (0,01-0,43) 0,005
G/A 1 8 20,0 (2,3-172,5) 0,005
A/A 0 0 NA NA
CD40 (rs6074022)
T/T 38 14 0,94 (0,41-2,19) 0,892
T/C 31 15 1,65 (0,71-3,17) 0,809
c/c 10 1 0,11 (0,01-1,85) 0,531
CD40 (rsll086998)
C/C 59 31 0,64 (0,06-6,55) 0,700
C/G 1 3 1,57 (0,15-16,15) 0,700
G/G 0 0 NA NA
Note: NA (not available) - the result is not presented because genotype/allele frequency is inadequate for a correct statistical analysis.
year, and, correspondingly, p=0,009). Male gender positively correlated with PR (rs=0.42, p=0.004) and was associated with a higher risk of a high MS PR (OR=2.21; CI 1.96 - 2.29; p=0.015). Therefore we analysed the associations of male or female genders with genotypes or alleles of the studied specificities. The correlation of a female gender and an allele A TNFRSF1A (rs4149584) with a higher
risk of a high MS PR was revealed (Table 3).
Earlier it was revealed that the risk of high MS PR is associated with certain characteristics of the MS onset in the population of MS patients in Altai Krai: MS onset age more than 27 years old, onset with motor disorders, duration of the first remission less than two years (Smagina I.V. et al. 2001a). These mechanisms were also confirmed in
Table 3
Relative Risk of a High Multiple Sclerosis Progression Rate Depending on the Combination of the Gender and Risk Alleles of the Specificities HLA-DRB1, TNFa (rs1800629), TNFRSF1A (rs4149584), CD40
(rs6074022, rs11086998)
Gene polymorphism, risk allele, gender Odds ratio, mean value (CI) Level of significance, P
HLA-DRB1(3), male NA NA
HLA-DRB1(3), female 0,49 (0,06-2,84) 0,365
HLA-DRB1(15), male 0,38 (0,05-2,84) 0,318
HLA-DRB1(15), female 0,52 (0,12-2,24) 0,367
TNFa (rsl800629) (allele A), male 3,61 (0,24-55,79) 0,315
TNFa (rsl800629) (allele A), female 1,31 (0,16-10,87) 0,787
TNFRSF1A (rs4149584) (allele A), male NA NA
TNFRSF1A (rs4149584) (allele A), female 2,78 (1,12-6,89) 0,026
CD40 (rs6074022) (allele C), male 0,38 (0,59-2,38) 0,276
CD40 (rs6074022) (allele C), female 0,27 (0,05-1,61) 0,144
CD40 (rsl1086998) (allele G), male NA NA
CD40 (rsl1086998) (allele G), female 0,47 (0,03-8,21) 0,588
Note: NA (not available) - the same as in Table 2
this study (Table 4). No correlation of the MS onset characteristics of poor prognosis with the studied gene polymorphisms was revealed.
2. Malkova N.A., Iyerusalimsky A.P. Multiple Sclerosis. Novosibirsk: NGMU; 2006.
3. Smagina I.V., Yelchaninova S.A., Zolovkina
Table 4
Relative Risk of a High Multiple Sclerosis Progression Rate Depending on the Disease Onset Characteristics
Parameter Odds ratio, mean value (CI) Level of significance, P
Onset age more than 27 years old* 2,37 (1,24-4,32) 0,004
Duration of the first remission less than 24 months* 3,54 (1,58-7,37) 0,002
Onset with pyramidal disorders 2,28 (1,16-4,66) 0,007
Onset with cerebellar disorders 2,04 (0,95-4,29) 0,015
Note: * - median is employed as a threshold value
The occurrence of MS relapses positively correlated with MS risk alleles - with the alleleA TNFa (rs1800629) and allele 3 HLA-DRB1 (Table 5)
The occurrence of MS relapses positively correlated with MS risk alleles - with the allele A TNFa (rs1800629) and allele 3 HLA-DRB1 (Table 5).
According to the odds ratio only HLA-DRB1*3 was associated with a higher risk of MS relapses more than once a year (OR=8.62; CI 1.25-59.72; p=0.027).
A.G., Gridina A.O. Factors Associated with the more rapid Progression of Multiple Sclerosis. Zhurn. nevrologii i psikhiatrii im. S.S. Korsakova Rasseyanny skleroz. 2011; 111 (2)(iss 2): 25-29. 4. Smagina I.V., Yelchaninova S.A., Zolovkina A.G. et al. Genetic Risk Factors for Multiple
Table 5
Correlation of the Risk Alleles of Specificities TNFa (rs1800629), TNFRSF1A (rs4149584), CD40 (rs6074022, rs11086998), HLA-DRB1 with the Occurrence of Multiple Sclerosis Relapses
Gene polymorphism, risk allele Spearman's rank correlation coefficient Level of significance, P
TNFa (rsl800629) (allele A) 0,205 0,040
TNFRSF1A (rs4149584) (allele A) - 0,177 0,076
CD40 (rs6074022) (allele C) 0,057 0,575
CD40 (rsll086998) (allele G) - 0,115 0,258
HLA-DRB1 (allele 15) - 0,115 0,256
HLA-DRB1 (allele 3) 0,194 0,042
According to the odds ratio only HLA-DRB1*3 was associated with a higher risk of MS relapses more than once a year (OR=8.62; CI 1.25-59.72; p=0.027).
Conclusion. G/A genotype of rs4149584 polymorphism of a TNFRSF1A gene coding the structure of cytokine receptors in the group of tumour necrosis factor alpha was associated with a high MS PR. This association reveals itself regardless of such predictors of a high MS progression rate as: onset age more than 27 years old, onset with motor disorders, duration of the first remission less than two years. The susceptibility to frequent occurrence of MS relapses significantly correlates with HLA-DRB1*3 allele that was earlier identified as the MS risk allele. The study shows that the results of HLA-DRB1, TNFRSF1A (rs4149584) genotyping can be used in the full assessment of risk factors of the unfavourable MS course in order to specify the management of these patients.
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Altai State Medical University.
Tel.: (3852) 689702.
Email: [email protected]
